Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.     

Superantigen gene profiles,genetic relatedness and biological activity of exosecretions of Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis

This study evaluated the superantigen gene profiles, genetic relatedness and biological activity of exosecretions of 50 Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis. Genomic relatedness of S. aureus was determined by pulsed field gel electrophoresis analysis of macro‐restricted chromosomes. The presence of genes encoding superantigens was confirmed by multiplex PCR. To study the biological activity of S. aureus exosecretions, the supernatants from bacterial liquid cultures were classified into three groups: those with leukotoxin‐like properties, those with superantigen‐like properties and those with no particular activity on leukocytes cultured in vitro. It was shown that all analyzed bacterial isolates belonged to the same clonal type and harbored the same combination of superantigen genes, namely sed, selj and ser. However, 22% of all isolates produced factors with superantigen‐like and 48% of them with leukotoxin‐like activities. Finally, although there were no detectable genetic differences between the analyzed bacterial isolates, the virulence factors secreted by them differed considerably.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

Quantitative trait loci for clinical mastitis on chromosomes 2, 6, 14 and 20 in Norwegian Red cattle

Mastitis is the most frequent and costly disease in dairy production and solutions leading to a reduction in the incidence of mastitis are highly demanded. Here a genome-wide association study was performed to identify polymorphisms affecting susceptibility to mastitis. Genotypes for 17 349 SNPs distributed across the 29 bovine autosomal chromosomes from a total of 2589 sires with 1 389 776 daughters with records on clinical mastitis were included in the analysis. Records of occurrence of clinical mastitis were divided into seven time periods in the first three lactations in order to identify quantitative trait loci affecting mastitis susceptibility in particular phases of lactation. The most convincing results from the association mapping were followed up and validated by a combined linkage disequilibrium and linkage analysis. The study revealed quantitative trait loci affecting occurrence of clinical mastitis in the periparturient period on chromosomes 2, 6 and 20 and a quantitative trait locus affecting occurrence of clinical mastitis in late lactation on chromosome 14. None of the quantitative trait loci for clinical mastitis detected in the study seemed to affect lactation average of somatic cell score. The SNPs highly associated with clinical mastitis lie near both the gene encoding interleukin 8 on chromosome 6 and the genes encoding the two interleukin 8 receptors on chromosome 2.     

Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing

Aims: Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next‐generation sequencing 454 GS‐FLX technology to elucidate the microbial community structure of cattle milk. Methods and Results: Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web‐based tool MG‐RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt–zinc–cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed. Conclusions: The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows. Significance and Impact of the Study: The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.     

Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus

Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine.     

The effect of out-wintering pad design on dirtiness score, somatic cell score and mastitis incidence in dairy cows

This study aimed to compare three woodchip out-wintering pad (OWP) designs, and indoor cubicle housing with regard to cow dirtiness scores during the winter housing period, and udder health during both the winter period and the following lactation, for spring-calving dairy cows. The treatments were: an uncovered (UP) and covered (CP) OWP with a concrete feed apron; an uncovered OWP with self-feed silage pit provided directly on the woodchips (SP); and indoor cubicle housing (IC). Data were compared during 2 years: year 1 was a case study while year 2 was an experimental study. In year 1, treatments were UP (space allowance = 12 m2/cow), CP (6 m2/cow) and IC. In year 2, all three OWP designs (12 m2/cow) were compared with IC. Animals were assigned to treatments at the end of lactation in the autumn, and remained there while dry until calving the following spring. Subsequently, all cows were at pasture during lactation. Outcome measures for analysis were cow dirtiness score, somatic cell score (SCS) and incidence of clinical mastitis during the dry period and during lactation. Quarter milk samples were also taken at drying off, calving and 3 weeks post partum both years, and at approximately 113 days in milk in year 2. Samples were analysed for presence of mastitis-causing agents and SCS was determined. Sub-clinical mastitis was diagnosed when cows had an SCS greater than 200 000, or California mastitis test greater than 1 in at least one quarter. In year 1, cows in CP were dirtier than cows in the other two treatments. These animals also had the highest SCS during lactation and tended to have more mastitis-causing agents isolated from quarter milk samples. In year 2, when all cows were stocked at the same density, cows in the sheltered OWP (i.e. CP) had similar dirtiness scores to cows in cubicles and significantly lower dirtiness scores than cows in the unsheltered OWP designs, i.e. UP and SP. However, there were no effects on SCS or quarter sample results. Cleaning of OWP's stocked at 12 m2/cow reduced cow dirtiness scores. However, cleaning of CP in year 1 when cows were stocked at 6 m2/cow had no effect on dirtiness scores. We conclude that dry cows stocked at 12 m2/cow on OWP's are unlikely to have udder health problems in the subsequent lactation. Furthermore, provision of shelter and cleaning of the woodchips are management factors that help to keep cows clean on OWP's.